The commercial exploitation of plants using genetic engineering has been an industrial goal for over a decade. Conventional approaches to the regulation of plant transgene expression by the fusing of a highly expressed promoter element directly with the gene coding sequence has proved insufficient to meet the stringent safety and technical demands of plant biotechnology today. Environmentally there is a serious risk of genetically releasing an actively expressed trait, such as herbicide resistance, into plant populations. Commercially the exploitation of plants by transgenic modification, as described for example by Koning et al (Plant Mol. Biol. Vol 18, pages 247-258 (1992)), such as through the introduction of a novel biochemical pathway such as polyhydroxybutyrate synthesis (described in WO92/19747 (ICI)) is hampered by the inability to introduce and coordinately regulate multiple transgenes in transgenic crops. The conventional approach would involve fusing each biosynthetic gene to a common promoter element, followed by their repeated transformation into a transgenic plant, as described for example in WO92/19747 (ICI). Practically, this approach is time-consuming, limits further alterations of transgene expression and rather than enabling coordinate transgene expression can lead to cosuppression of other transgenes, Meyer P. and Saedler H, Ann. Rev. Plant Physiol. Plant. Mol. Biol., 1996, vol. 47, pages 23-48, and Baulcombe D. C. and English J. J., Curr. Opin. Biotech., 1996, vol. 7, pages 173-180).
The present invention is directed to a novel approach to the control of transgenes in plants. Instead of using the regulatory and expressed sequences in conventional cis fashion, the present invention rearranges the regulatory and expressed sequences so that they are used in a trans fashion.
The promoter element now indirectly regulates the transgene(s) via a transcriptional activating protein intermediate. The immediate outcome of this is that two plant lines can be produced; one which contains transgene(s) encoding for a desired phenotype and one which contains a transgene encoding for the regulatory transactivating protein. This means that the desired phenotypic trait is only fully expressed once both of the sets of transgenes have come together in an F1 hybrid plant. The regulatory and phenotype transgenes will then segregate apart in subsequent generations.
This has major safety implications because it means that the chances of an active transgene encoding for a phenotypic trait, such as herbicide resistance, being released into the environment is considerably reduced. Safety can be further enhanced by making one of the plants containing the transgenes male sterile, so that pollen contain the transgene is not released. This also has advantages for seed companies marketing high value genetically engineered traits because, if a farmer attempts to use F2 generation seed, he will see a dramatic reduction in the amount of product produced by the F2 generation plants.
The use of a transcriptional protein also has the added advantage that several transgenes can be controlled by the same transactivating protein, without the problems of cosuppression seen with conventional cis acting systems.
By physically separating the promoter regulatory and target sequences within independent transgenic plants, different transgene expression can be selected for in the F1 generation simply by crossing the transgenic target line with regulator lines that express the transactivating protein in particular spatial and temporal patterns (e.g. seed or leaf). Hence, this system allows the rapid introduction and fine-tuning of commercially attractive single or multiple gene traits in transgenic crops.
It also allows a useful tissue specific, but weakly expressed promoter to be used, since the transactivating protein works in low concentrations.
The preferred transactivating protein used is GAL4 from the yeast, Saccharomyces cerevisiae or ArgR from E-coli.
The expression of genes encoding enzymes of the galactose and melibiose metabolic pathways in the yeast Saccharomyces cerevisiae is stringently regulated by the available carbon source (Johnston, Microbiol. Rev., Vol. 51, pages 458-476 (1987)). Transcriptional control is mediated through the positive regulatory protein GAL4 and the negative regulatory protein GAL80. In the presence of galactose GAL4 divergently promotes transcription of the genes of the galactose regulon. Transcriptional activation by GAL4 results in a 1,000 fold increase in the level of gene expression. When the inducer is absent GAL80 inhibits the transactivating ability of GAL4. A number of additional transcriptional control mechanisms operate in the presence of glucose. These mechanisms, collectively termed catabolite repression, ensure that glucose is the preferred carbon source.
Native GAL4 is 881 amino acid (aa) residues in length and has a molecular weight of 99,000. Deletion and domain swap analyses have demonstrated that GAL4 is comprised of a number of functionally delineated domains, the combined activities of which account for the protein's in vivo characteristics (Ma & Ptashne, Cell. Vol. 48, pages 847-853, (1987); Brent & Ptashne, Cell. Vol. 43, pages 729-736, (1985). GAL4 binds to a 17 base-pair (bp) sequence exhibiting dyad symmetry, termed the galactose upstream activating sequence (UAS.sub.G). In the presence of galactose GAL4 activates expression of genes linked to the UAS.sub.G (West et al., Mol. Cell. Biol., Vol. 4, pages 2467-2478) (1984). A consensus sequence of the naturally occurring site will also mediate GAL4 stimulatory action (Giniger et al., Cell, Vol. 40, pages 767-774, (1985); Lord et al., J. Mol. Biol., Vol. 186, pages 821-824 (1985). The amino terminal (N-terminal) 65 aa residues of GAL4 are responsible for sequence specific-binding (Keegan et al., Sci. Vol. 231, pages 699-704 (1986); Johnston, Nature, Vol. 328, pages 353-355 (1987). Sequence-specific binding is absolutely dependent on the presence of a divalent cation coordinated by the 6 cysteine residues present in the DNA binding domain. The zinc-containing domain recognizes a conserved CCG triplet at the end of each 17 bp site via direct contacts with the major groove (Marmorstein et al., Nature. Vol. 356, pages 408-414 (1992). Each target DNA sequence binds GAL4 as a dimer (Carey et al., J. Mol. Biol. Vol. 209, pages 423-432 (1989), a function ascribed to aa residues 65-94. Also present in the N-terminal 1-78 aa residues is a nuclear localization sequence (Silver et al, PNAS (USA), Vol. 81, pages 5951-5955 (1984).
Binding of GAL4 to its target DNA sequence is insufficient to direct RNA polymerase II dependent transcription of linked genes. The DNA binding function of the protein serves solely to position the carboxy-terminal (C-terminal) transcriptional activating domains in the vicinity of the promoter. Transcriptional activation is conferred by 2 major activating domains termed activating region I (ARI-aa residues 148-196) and activating region II (ARII-aa residues 767-881), of which ARII is the more potent (Ma & Ptashne, Supra). A third cryptic transactivating region (aa residues 75-147) has been identified in GAL4 deletion derivatives and exhibits in vitro activity (Lin et al., Cell. Vol. 54, pages 659-664 (1988). Each of the three transcriptional activation domains is characterized by a high proportion of negatively charged aa residues and hence are known as acidic activation domains (AAD). In the absence of a DNA-binding domain the activating regions are unable to function.
The mechanisms responsible for eukaryotic transcriptional activation have been evolutionary conserved. This is indicated by the fact that the yeast transcriptional activator GAL4 can activate gene expression in other eukaryotic organisms. Native GAL4 has been demonstrated to activate transcription of genes linked to the GAL4 binding site (either synthetic or the UASG) in insect (Fischer et al. (1987) and mammalian cells (Kakidani & Ptashne, Cell. Vol. 52, pages 161-167 (1988); Webster et al., Cell. Vol. 52, pages 169-178 (1988). Full length GAL4 is, however, incapable of stimulating transcription in plant protoplasts possibly as a result of its inefficient synthesis or instability (Ma et al., Nature, Vol. 334, pages 631-633 (1988). Deletion derivatives of GAL4 are able to activate transcription in yeast. These proteins, comprised of aa residues 1-147 (DNA-binding domain) and ART and/or ARII also exhibit activity in mammalian cells (Kakidani & Ptashne, Supra) and plant protoplasts (Ma et al., Supra).
ArgR is the arginine repressor from Escherichia coil. The action and isolation of ArgR is reviewed in the article by Werner K Maas (Micorbiol. Reviews, 1994, Vol. 58 (4), pages 631-640), incorporated herein by reference.
The product of the ArgR gene, in conjunction with L-arginine, controls the synthesis of the 10 enzymes of arginine biosynthesis and also its own synthesis. The ArgR gene product in its native form acts as a repressor of transcription. The 12 genes of the arginine regulon are organized into nine transcriptional units, each containing an operator site overlapping a promotor. An operator site consists of two 18-bp palindromic sequences referred to as ARG boxes to which the repressor binds.
The inventors realized that, whilst ArgR in its native state is a repressor, the ArgR DNA binding domain may be used to form chimera constructs with parts of other genes such as, for example the GAL 4 activation domain II, which may be used as transgene activators.
Two recent reports have demonstrated transgene expression of a target gene arranged in trans with a control gene in Drosophila (Brand & Perrimon, Development, Vol. 118, pages 401-415 (1993) and Crieg & Akam, Nature, Vol. 362, pages 630-632 (1993)). Neither of these discuss the possibility of using such a system in plants.
The system described herein can be used to control the production of products or of a desired trait such as herbicide resistance. A preferred multigene system is the use of genes involved in the biosynthesis of polyhydroxybutyrate (PHB), controlled by a transactivating protein.
PHB is a commercially important biodegradable polymer which has previously been produced in plants using conventional cis acting control, as described in WO92/19747 (ICI) and Pimer et al (Science, Vol 256, pages 529-523 (1992)). It is, however, an ideal product to be produced by the invention since the multigene pathway is subject to the problems of cosuppression when used in conventional systems and the trans regulating system described herein enables the PHB to be produced more safely than existing methods of producing its implants.
Other genes which may be controlled include genes for controlling male sterility, such as the ribonuclease barnase and its inhibitory subunit barstar (Mariani C. et al, Nature, 1990. vol. 347, pages 737-741). Antisense or sense RNA mediated inhibition of target mRNAs such as polygalactonuronidase and ACC oxidase during tomato ripening may also be controlled by the transgene system of the invention (Smith C. J. et al. Nature, 1988, vol. 334, pages 724-726 and Hamilton A. J. et al. Nature, 1990, vol. 346, pages 284-287).
It is therefore an object of the invention to produce an inherently safe method of producing a phenotypic trait in transgenic plants.
It is another object to produce a method of regulating two or more genes in a plant without the problems of cosuppression of the genes associated with conventional methods.